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ORIGINAL ARTICLE Table of Contents  
Ahead of print publication
Association of red complex bacteria with periodontal disease: A clinico microbiological study


1 Department of Periodontics, Institute of Dental Sciences Sehora, Jammu, Jammu and Kashmir, India
2 Himachal Institute of Dental Sciences, Himachal Pradesh, India

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Date of Submission05-Mar-2021
Date of Decision10-Apr-2021
Date of Acceptance07-Jun-2021
Date of Web Publication05-Jul-2021
 

  Abstract 


Context: Oral cavity offers diverse habitats to various species of different microorganisms; some of them, under certain conditions cause loss of connective tissue and bone, ultimately leading to periodontitis. Once such complex of bacteria often referred to as red complex bacteria consisting of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been regarded as those associated with periodontitis. With increasing advances in the field of medicine N-benzoyl-DL-arginine-2-naphthylamide (BANA) test, a modern chair-side paraclinical method has been designed to detect the presence of one or more such bacteria. Aim: The aim of the study was to detect the presence of these bacteria in patients suffering from various forms of periodontitis using BANA test kit. Settings and Design: A total of 1500 patients suffering from various forms of periodontitis were selected for the study. Subjects and Methods: These were divided into three groups: Group I: Mild periodontitis, Group II: Moderate periodontitis, and Group III: Severe periodontitis. Microbial parameters were recorded using BANA test. Statistical analysis used Mann–Whitney test and Pearson's Chi-square test. Results: Group III had significantly greater percentage of BANA-positive sites followed by Group II and Group I. Conclusions: There is a positive correlation between the presence of red complex bacteria and severity of periodontitis.

Keywords: Dental plaque, N-Benzoyl-DL-arginine-2 naphthylamide, oral microbiota, periodontal disease, red complex bacteria


How to cite this URL:
Singh M, Sahota JK, Singh P, Kour H. Association of red complex bacteria with periodontal disease: A clinico microbiological study. Apollo Med [Epub ahead of print] [cited 2021 Nov 30]. Available from: https://www.apollomedicine.org/preprintarticle.asp?id=320721





  Introduction Top


Oral cavity contains microorganisms of different species which aggregate and prosper and this is termed as “dental plaque.” These microbes then adhere to tooth surface and multiply in shielded environment. As proposed by Socransky et al., most of the pathogen of all complexes comprise Porphyromonas gingivalis (Pg), Tannerella forsythia, and Treponema denticola (Td) also collectively known as Red Complex. These red complex bacteria have shown to be most important periodontal pathogens in causing periodontal disease[1] by production of various trypsin-like enzymes that degrade intercellular matrix of periodontal tissues.[2] With advances in technology, the detection of the presence of these microorganisms often becomes a matter of grave concern for clinicians. N-benzoyl-DL-arginine-2-naphthylamide (BANA) enzymatic test is a rapid and reliable chairside diagnostic test, which can be performed in about 15 min time and can give information about the presence of three these putative pathogens in subgingival plaque samples. The aims of this study were to assess the correlation between presence of red complex bacteria and severity of periodontitis using BANA test kit.

Principle of N-benzoyl-DL-arginine-2-naphthylamide test

Anaerobic bacteria contain enzymes that can hydrolyze peptides but only P. gingivalis, T. denticola, and Tanerella forsythia possess significant amount of a unique enzyme. Taking advantage of these unique characteristics, researchers at the University of Michigan developed a synthetic peptide, BANA to detect the presence of the shared enzyme. The BANA-Zyme™ reagent strips are plastic cards to which separate reagent-containing matrices are affixed. The lower matrix is impregnated with BANA. Subgingival plaque samples were applied to the lower matrices. The upper reagent matrix contains a chromogenic diazo reagent (fast black K& #43;). Peptidase in certain anaerobic microorganisms associated with periodontal diseases can hydrolyze the peptide analog BANA. The upper reagent matrix, which reacts from BANA by bacterial enzyme, reacts with fast black K+ forming a permanent blue color. The blue color of a positive or weak positive reaction appears in the upper matrix and is permanent. Blood and saliva do not hydrolyze BANA and do not interfere with the test, but blood in the sample may obscure the visualization of the blue color. The processor is a device, in which strips are placed which then changes the color of sample to show the presence or absence of bacteria.


  Subjects and Methods Top


The present study was carried out in the Department of Periodontology and Oral Implantology of our Institute and the study was approved by our Institute ethical committee vide no: Ethics/Perio/Oct/2019. Patients suffering from different form of periodontitis belonging an age group of 35–75 years and having bilateral pockets of 5–7 mm in molars were selected for the study. Those patients who presented themselves with any systemic disease or were undergoing any local or systemic antimicrobial and anti-inflammatory therapy for the past 6 months or periodontal therapy other than standard prophylaxis during previous 6 months were excluded from the study. Pregnant, lactating women, tobacco chewers, and alcoholics were also excluded from the study.

Study design

A simple randomized, clinical study was conducted to check the presence of red complex bacteria in periodontitis patients with various degrees of severity. A total of 1500 patients with 3000 sites were randomly divided into following three groups based on case definition and classification given by Prevention and American Academy of Periodontology [Table 1][3],[4] into:
Table 1: Prevention (case definition and classification) and American academy of periodontology

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  1. Group I: Patients suffering from mild periodontitis
  2. Group II: Patients suffering from moderate periodontitis
  3. Group III: Patients suffering from severe periodontitis.


Each category contained equal number of patients, i.e., 500 each. The nature and design of the clinical study were explained, and informed consent was obtained from all the participants and BANA chair side test was performed.

Procedure

Subgingival plaque sample from each patient was obtained using curette and was applied on to the raised reagent matrix affixed to the lower portion of the test strip. The upper test strip was moistened with distilled water using a cotton swab. BANA test strip was folded at the given crease mark so that the lower and upper reagent strips were meet. It was then placed into either of the slots on the top of the processor. The heating element of the processor started automatically when strip was inserted into the bottom of the slot, as indicated by the flashing light. The test strips color development was completed when the indicator light went off and the bell rang and was removed from the processor, and then the lower reagent strip was discarded to prevent the contamination of it further. The upper reagent strip was examined for the presence of blue color. If a blue color was a detected, the site was marked as either weak positive or positive [Figure 1]. Recording was done for each sampled site as negative, weak positive, or positive.
Figure 1: Colour change after performing BANA test indicative of presence of r ed complex bacteria

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  Results Top


All patients (950 males and 550 females with mean age of 53 ± 5 years) completed the study. The darkness of blue color ranged from deep blue (for patients with severe periodontitis) to pale blue (for patients with moderate periodontitis). However, as done for the patients of gingivitis, the color ranged from light yellow to salmon color depending on the severity of gingivitis indicating the test to be weak negative as red complex bacteria were not detected in the same group. Recordings were carried out on the 1st day before treatment. All recordings were subjected for statistical analysis using Mann–Whitney and Pearson Chi-square test. Microbiological assessment between the groups showed significance [Table 2]. However, it showed that BANA is sensitive but has low specificity as it could only determine the presence of red complex bacteria but could not specify the type of bacteria and the levels of individual bacteria could not be judged after performing BANA test.
Table 2: Pearson's Chi square test for Group I,II,III

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  Discussion Top


Periodontitis is an infectious disease caused by bacteria, brings about destructive changes leading to loss of bone and connective tissue attachment. Several oral bacteria such as the black-pigmented, Gram-negative anaerobic rods P. gingivalis, T. forsythia, and T. denticola have been considered as a major pathogen in periodontitis.[5] These bacteria were most crucial for the progression of this disease, and thus their detection provided essential information about the severity of periodontitis. A diagnostic test should be useful, ideally leading to a choice of treatment that would confer benefits upon the patient. It should be simple and aimed at identifying certain forms of periodontal disease associated with some predominant periodontopathogens, which possess few common characteristics. Currently, there are many methods directed at identifying periodontal pathogenic species: Microbial cultures, DNA probes, and polymerase chain reaction. The latest two methods can detect uncultivable species, but they require good laboratory equipment and they cannot be used as routine tests. Another category of tests includes the chair-side tests, biochemical, and enzymatic tests. BANA test is a modern chair-side paraclinical method designed to detect the presence of one or more anaerobic bacteria commonly associated with periodontal disease, namely T. denticola, P. gingivalis, and Tannerella forsythus in subgingival plaque samples taken from periodontally diseased teeth. The BANA test was developed by Dr. Walter Löesche and coworkers at Michigan University, being the result of more than 15 years of research, and they proposed the use of BANA reaction to detect the presence of periodontal pathogens and thus serve as a reliable marker of disease activity and also aid in monitoring periodontal therapy. The study was conducted in 1500 patients to evaluate the association of red complex bacteria with severity of periodontal disease in patients suffering from chronic periodontitis. The microbiological assessment was performed using a chairside BANA test showing specific microbiota. P. gingivalis, T. forsythia, and T. denticola species share a common enzymatic profile and have a trypsin-like enzyme in common. This activity of enzyme can be measured with the hydrolysis of the colorless substrate BANA; hence, all the three BANA-positive species are frequently cited as potential periodontal pathogens.[6] In our study, we found out that there was a positive correlation between the presence of red complex bacteria and the severity of periodontitis. The percentage of BANA-positive sites was significantly higher for Group III followed by Group II and Group I [Graph 1], [Graph 2], [Graph 3]. This was attributed to the presence of more red complex bacteria in periodontal pockets which clinically showed more periodontal destruction in severe periodontitis as compared to moderate and mild form. The ability of BANA to detect a particular threshold of anaerobic periodontopathic bacteria was found to be a valuable diagnostic tool for screening the individual at risk for an anaerobic infection and also as an objective indicator of periodontal disease activity that could be used in combination with the clinical criteria both to initiate therapy and as a means to monitor the efficacy of treatment.




  Conclusion Top


There is a positive correlation between the presence of red complex bacteria and the severity of periodontitis and BANA test can be used as a reliable indicator of BANA-positive species in dental plaque.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr, Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25:134-44.  Back to cited text no. 1
    
2.
Slots J, Bragd L, Wikström M, Dahlén G. The occurrence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius in destructive periodontal disease in adults. J Clin Periodontol 1986;13:570-7.  Back to cited text no. 2
    
3.
Page RC, Eke PI. Case definitions for use in population-based surveillance of periodontitis. J Periodontol 2007;78 Suppl 7S:1387-99.  Back to cited text no. 3
    
4.
Eke PI, Page RC, Wei L, Thornton-Evans G, Genco RJ. Update of the case definitions for population-based surveillance of periodontitis. J Periodontol 2012;83:1449-54.  Back to cited text no. 4
    
5.
Suzuki N, Yoneda M, Hirofuji T. Mixed red complex bacterial infection in periodontitis. Int J Dent 2013;1:1-5..  Back to cited text no. 5
    
6.
Amalfitano J, De Filippo AB, Bretz WA, Loesche WJ. The effects of incubation length and temperature on the specificity and sensitivity of the BANA (N-benzoyl-DL-arginine-naphthylamide) test. J Periodontol 1993;64:848-52.  Back to cited text no. 6
    

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Correspondence Address:
Malvika Singh,
Department of Periodontics, Institute of Dental Sciences Sehora, Jammu, Jammu and Kashmir
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/am.am_19_21



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    Tables

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    -  Singh M
    -  Sahota JK
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    -  Kour H


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